Glycosylceramides perform important immunological functions as T cell antigens presented by CD1, a family of lipid-binding MHC-like molecules. For example, an alpha-galactosylceramide (alphaGC) specifically activates regulatory NKT cells, which modulate, through the release of either Th1 or Th2 cytokines, various disease conditions. Other glycosylceramides may elicit adaptive T cell responses involved in Multiple Sclerosis. Thus, the program project uses glycosylceramides as a model antigen family to study fundamental issues of glycolipid antigen presentation by CD1d molecules with potential clinical applications. A multidisciplinary approach encompassing glycolipid synthetic chemistry, protein expression, biophysics and structural biology, as well as cellular immunology and cell biology, has been assembled. Synthetic variants of alphaGC, the NKT cell antigen, will be generated to study the structural determinants of their adjuvant properties. We will use new approaches to visualize the cellular uptake and trafficking, CD1 loading and TCR binding of glycosylceramides and will search for new variants with selective Th1 or Th2 adjuvant properties modulating expression of diseases such as type I diabetes, experimental encephalomyelitis and cancer. In addition to the innate NKT cell response to alphaGC, we also will probe the diversity and specificity of the glycolipid-specific adaptive TCR repertoire, a prerequisite to design glycolipid-specific T cell vaccines. The individual research projects are as follows. Project 1, Savage: Design of alphaGC structural variants with selective Th1 or Th2 properties. Project 2, Teyton: Biophysical and structural aspects of glycosylceramide interaction with CD1d and TCR. Project 3, Bendelac: T cell repertoire and functions associated with glycosylceramide recognition in vivo in mice and intracellular trafficking properties of glycosylceramides.